One of the families of cellular oncogenes most commonly implicated in a wide variety of malignancies is the c-ras family. Overexpression of the c-ras gene has been observed in a human colon cell line (McCoy et al, Nature London 32, 79-81, 1983). Thus, both quantitative and qualitative changes affecting the level of expression of the c-ras genes may be considered to be associated with tumorigenesis.
Genes related to ras have been identified in lower organisms such as Saccharomyces cerevisiae, Drosophila and Dictyostelium. The presence of ras genes in nocancerous cells and in evolutionarily diverse organisms suggested that these genes have fundamental importance in cellular physiology. Saccharomyces cerevisiae contains six genes whose protein products share homology with the mammalian ras oncogene products. Yeast ras-related protein YP2 shares about 40% homology in the amino terminal 160 amino acids with mammalian ras proteins (Gallwitz et al., Nature London 306, 704-7, 1983). Furthermore, RAS1 and RAS2 yeast genes are 62% homologous with mammalian ras in the same region (Dahr et al, Nucl. Acids Res. 12, 3611-18, 1984 and Powers et al Cell 36, 607-12, 1984).
Also, two rho genes with significant homology to the ras oncogene have been isolated recently (Maduale et al, Proc. Natl. Acad. Sci. 84, 779-89, 1987). Another gene, SEC4 was recently shown to share 32% homology with ras proteins (Salminen et al. Cell 49, 527-38, 1987).
A description of the ras gene family, on which the present invention does apply is given in a recent review article (M. Barbacid, Ann. Rev. Biochem, Vol 56, 779-827, 1987). These c-ras genes are expressed at low level in most cells. Cross reactive antisera have detected low levels of p21 proteins in almost all cell lines examined (Langbeheim et al, Virology 106, 292-300, 1980). However, the transcriptional activity of ras oncogenes (Ha-ras, Ki-ras) was found to be greater in malignant than in normal tissues. This included renal cell carcinoma, ovarian and colon adenocarcinoma, carcinoma of the lung and the breast and acute myeloid leukaemia (Slamon et al. Science 224, 256-62, 1984).
In a previous abstract paper (Baratal and Shalitin, Proc. Amer. Assoc. for cancer Res. 27, 427, 1986) we described the generation of anti-YP2 rabbit polyclonal antibodies against native yeast ras related protein YP2. These antibodies were shown to immunoreact with mammalian p21protein.
As known all members of the ras gene family encode closely related proteins having a molecular weight of about 21,000 daltons, which have been designated p21. The level of p21 expressions is similar in many different human tumor cell lines, independent of whether the cell line contains an activated ras gene detectable by transfection (Der and Cooper, 1983, Cell 32, 201-208).
Human neoplasia could be determined by histological examination of by DNA-mediated transfection assay with mouse NIH 3T3 fibroblasts. Foci of transformed cells are scored after 21 days and only 10% of the urinary tract tumors yielded foci in this test (Fujita et al., 1984, Nature 309, 464-466).
Nucleotide sequence analysis of the ras.sup.H transforming gene of human bladder carcinoma cells indicated that the transforming activity of this gene is a consequence of a point mutation altering amino acid 12 of p21 from glycine to valine (Tabine et al. 1982, Nature 300. 143-149; Reddy et al., 1982, Nature 300, 149-152; Capon et al., 1983, Nature 302, 33-37; Reddy, 1983, Science 220, 1061-1063.)
The altered p21 protein displayed abnormal electrophoretic mobility on SDS-polyacrylamide gels. Furthermore, proteins encoded by ras.sup.K genes previously activated in four human lung and colon carcinoma cell lines, also displayed abnormal electrophoretic mobilities (Der and Cooper, 1983, Cell 32, 201-208). It further appeared that different mutations could activate the same rsa.sup.K gene in different individual neoplasms (Der and Cooper, 1983, Cell 32, 201-208).
Detection of human cancer cells by molecular diagnosis of restriction sites is tiresome. An easy way of diagnosis is by electrophoretic separation of proteins derived from cancer cell extracts on SDS - polyacrylamide gels, (Western blots) and staining by specific antibodies. Carcinoma cells which express ras proteins with identical electrophoretic mobility to those of normal human cells, might express the protein at a level 3-5 fold higher than primary fibroblasts or epithelial cultures (Der and Cooper, 1983, Cell 32, 201-208).
It is an object of the present invention to provide anti p20 antibodies which are very specific in their binding properties towards human p21 Ha-ras and Ki-ras gene products using immunoprecipitation, immunoblotting and indirect immunofluorescence.